Recent studies indicate a causative link between defects in autophagy, a lysosome-dependent intracellular degradation pathway, and Parkinson's disease (PD). Conversely, up-regulation of autophagy has been proposed as a possible PD intervention strategy. We recently identified USP24, a gene of unknown function located in the PD associated PARK10 locus, as a negative regulator of autophagy. Our data indicate that USP24 protein levels are increased in the substantia nigra of a subpopulation of PD patients, suggesting possible involvement also in idiopathic PD. On the other hand, inactivation of USP24 induces autophagy and can play a protective function in cellular models of PD. The goal of our proposal is to use in vitro PD models to test the hypothesis that USP24 affects PD predisposition via autophagy-dependent mechanism and that inhibition of USP24 can attenuate PD pathology. AIM 1 will determine how USP24 overexpression and PD-associated coding region polymorphisms alter levels of autophagy. We hypothesize that USP24 inhibits autophagy flux by interfering with function of the type III PI3 kinase. We will test this by expressing wild-type and mutant USP24 in human cell lines and primary rat neurons and using immunofluorescence, western blot and autophagic flux analysis to investigate the effects on the levels of autophagy, type III PI3 kinase activity, lysosomal degradation and mitochondrial function. AIM 2 will determine the influence of USP24-regulated autophagy on outcomes in cell based PD models. We hypothesize that induction of autophagy by USP24 inhibition will attenuate PD pathology. To test this we will evaluate the effects of USP24 knock-down, overexpression and PD polymorphisms on cell viability, mitochondrial function and protein turnover in two cell based PD models: treatment with PD associated herbicide paraquat and expression of mutant alpha-synuclein A53T. Our data will be the necessary first step to understand how USP24 regulates autophagy and contributes to PD and to determine if up-regulation of autophagy by inhibition of USP24 can be used as a novel prevention and treatment strategy.